Parkinson's disease is a common neurodegenerative disorder that results from degeneration of dopamine (DA) neurons in the nigro-striatal system. Transplantation of fetal DA neurons can relieve Parkinsonism in some patients; however, limited tissue supply is a major obstacle for widespread use of fetal cells. Human embryonic stem (hES) cells could provide the platform for creating an unlimited supply of human DA neurons for cell therapy of Parkinson's disease. The goal of this study is to develop DA neurons from hES cells (NIH registration code ES01-06) and to demonstrate their function and therapeutic potential in animal models of Parkinson's disease. We have recently developed highly-enriched (>95 percent) cultures of expandable, developmentally competent neural progenitors (NPs) from hES cells. The NPs differentiate spontaneously into neurons expressing tyrosine hydroxylase (TH), however, at a low frequency. Our preliminary data suggest that defined signals can significantly promote the differentiation of hES cell-derived NPs towards TH+ neurons. In this study we will further develop the protocols to direct the differentiation of hES cells into TH+ neurons by the following approaches: (A) Administration of growth factors and cytokines that are known to induce a midbrain fate. (B) Forced expression of key transcription factors in the development of DA neurons. (C) Co-culture with stromal cells that have DA fate-inducing activity. Potential synergism between the strategies will be determined. We will evaluate whether hES cell-derived TH+ neurons have electrophysiological and functional properties expected from midbrain DA neurons and whether they can lead to recovery in the rat model of Parkinson's disease. Our preliminary results suggest that transplantation of hES cell-derived NPs to the DA-depleted striatum of rats results in behavioral recovery of DA-mediated motor asymmetry. Lastly, we will evaluate the potential of hES cell transplantation to correct behavioral deficits and the abnormal electrical activity of basal ganglia neurons in the MPTP primate model, which most reliably mimics the human disorder. This study will pave the way for further developments that may eventually allow the use of human ES cells as an unlimited source of midbrain neurons for transplantation in Parkinson's disease.